Human parainfluenza virus type 1 (HPIV1) is a significant cause of severe respiratory tract disease in infants and young children. HPIV1 is an enveloped, non-segmented, single-stranded, negative-sense RNA virus belonging to the subfamily Paramyxovirinae within the Paramyxoviridae family, which also includes the HPIV2 and HPIV3 serotypes. These serotypes can be further classified as belonging to either the Respirovirus (HPIV1 and HPIV3) or Rubulavirus (HPIV2) genus and are immunologically distinct in that primary infection does not result in cross-neutralization or cross-protection. The HPIV1 genome encodes three nucleocapsid-associated proteins including the nucleocapsid protein (N), the phosphoprotein (P) and the large polymerase (L) and three envelope-associated proteins including the internal matrix protein (M) and the fusion (F) and hemagglutinin-neuraminidase (HN) transmembrane surface glycoproteins. F and HN are the two viral neutralization antigens and are the major viral protective antigens. The HPIVs cause respiratory tract disease ranging from mild illness, including rhinitis, pharyngitis, and otitis media, to severe disease, including croup, bronchiolitis, and pneumonia. HPIV1, HPIV2 and HPIV3 have been identified as the etiologic agents responsible for 6.0%, 3.3% and 11.5%, respectively, of hospitalizations of infants and young children for respiratory tract disease. Together these viruses account for approximately 20% of all pediatric hospitalizations due to respiratory disease. A licensed vaccine is currently not available for any of the HPIVs.[unreadable] HPIV1 vaccine development: We further sought to understand the role of the C proteins in HPIV1 replication in vitro and in vivo. Recombinant HPIV1 (rHPIV1) was modified to create rHPIV1-P(C-), a virus in which expression of the C proteins (C&#8242;, C, Y1 and Y2) was silenced without affecting the amino acid sequence of the P protein. Infectious rHPIV1-P(C-) was readily recovered from cDNA, indicating that the four C proteins were not essential for virus replication. rHPIV1-P(C-) replicated in vitro as efficiently as HPIV1 wt early during infection, but its titer subsequently decreased coincident with the onset of an extensive cytopathic effect (cpe) not observed with rHPIV1 wt. rHPIV1-P(C-) infection, but not rHPIV1 wt infection, resulted in activation of caspase 3 and caused nuclear fragmentation in LLC-MK2 cells, identifying the HPIV1 C proteins as inhibitors of apoptosis. In contrast to rHPIV1 wt, rHPIV1-P(C-) and rHPIV1-CF170S each induced IFN and did not inhibit IFN signaling in vitro. However, only rHPIV1-P(C-) induced apoptosis. Thus, the anti-IFN and anti-apoptosis activities of HPIV1 were separable: both activities are disabled in rHPIV1-P(C-) whereas only the anti-IFN activity is disabled in rHPIV1-CF170S. In AGMs, rHPIV1-P(C-) was considerably more attenuated than rHPIV1-CF170S, suggesting that disabling the anti-IFN and anti-apoptotic activities of HPIV1 had additive effects on attenuation in vivo. Thus, the C proteins of HPIV1 are non-essential but have anti-IFN and anti-apoptosis activities required for virulence in primates.[unreadable] In an in vitro model of human ciliated airway epithelium (HAE), a useful tool for studying respiratory virus-host interactions, HPIV1 wt selectively infected ciliated cells within the HAE and progeny virus was released from the apical surface with little apparent gross cytopathology. In HAE, type I IFN is induced following infection with rHPIV1-CF170S but not following infection with HPIV1 wt. IFN induction coincided with a 100 to 1000-fold reduction in virus titer, supporting the hypothesis that the HPIV1 C proteins are critical for inhibition of the innate immune response. [unreadable] A microarray-based analysis of the kinetics of gene expression of respiratory epithelial cells infected with wt or mutant HPIV1 or treated with IFN&#946; was performed to examine: 1) how wt HPIV1 infection alters human respiratory epithelial cell gene expression; 2) what role IFN&#946; plays in this response; 3) how the response to infection with the C mutant viruses, rHPIV1-CF170S and rHPIV1-P(C-), compares to infection with wt HPIV1; and 4) whether the phenotypic differences between the two C mutant viruses (level of attenuation and apoptosis phenotype) can be explained at the transcriptional level. mRNA levels in A549 cells treated with IFN&#946; or infected with HPIV1 wt, rHPIV1-CF170S, or rHPIV1-P(C-) were compared using a microarray that represented the full complement of known human genes. By 48 h post-infection, the CF170S mutant significantly induced 1,632 genes and suppressed 690 genes, whereas P(C-) induced 1,255 genes and suppressed 168 genes by 48 h p.i. Wt HPIV1 significantly induced 274 genes and suppressed 3 genes, and IFN&#946; treatment induced 176 genes and suppressed 1 gene. Therefore, rHPIV1s encoding C gene mutations modify the expression of up to 2,322 genes, whereas HPIV1 encoding the wt C gene modifies the expression of only 277 genes, indicating the profound effect the C proteins have on suppression of the host cell response to HPIV1 infection. The gene expression profiles of rHPIV1-P(C-) and rHPIV1-CF170S infected cells did not differ. In summary, the HPIV1 C proteins exert remarkable control over the cellular transcriptional response to viral infection, indicating that the C proteins are important virulence factors for HPIV1. Mutations within the C gene permit the activation of a broad array of cellular genes involved in the type I IFN, IRF3 and NF-kB pathways that would otherwise be repressed by HPIV1 infection, and these mutations specify an attenuation phenotype in vivo. [unreadable] HPIV2 vaccine development: Reverse genetics was used previously to generate attenuating mutations in the L polymerase protein of human parainfluenza virus type 2 (HPIV2) and to enhance their genetic stability. Last year we described the construction of two highly attenuated viruses, rHPIV2-V94(15C)/460A948L and rHPIV2-V94(15C)/948L/&#916;1724, that were immunogenic and protective against challenge with wild-type HPIV2 in African green monkeys. A clinical lot of rHPIV2-V94(15C)/948L/&#916;1724 has been manufactured and will be evaluated in humans in 2008 and 2009.[unreadable] The V gene of HPIV2, which has been described as an IFN antagonist promoting STAT2 degradation, is another target for the introduction of attenuating mutations. Since the P and V ORFs in the P/V gene of HPIV2 overlap, we separated the P and V ORFs into two gene units to permit the introduction of genetically stable mutations into V without affecting P. A HPIV2 P+V virus was recovered that had wt phenotype in vitro and in vivo. The V protein contains a RING finger-like domain in its C terminal region that is involved in STAT2 degradation. Mutations of the seven cysteine amino acid residues of the RING domain to serine were either lethal (could not be recovered) or highly debilitating, i.e., virus replication was over 1000-fold reduced in vitro, even in Vero cells that do not produce IFN. This indicated that the V protein is required for efficient replication of HPIV2 in vitro and that the RING domain is not a useful site for the introduction of att mutations intended for use in HPIV2 vaccines. We are seeking to identify mutations that modulate STAT2 degradation but that permit efficient replication in vitro.